Objective 1. Explore the use of a cocktail containing phages and predatory bacteria to kill Vibrio parahaemolyticus in market oysters. Objective 2. Compare and contrast Halobacteriovorax and phage levels in oysters, seawater, and sediment as a prerequisite to the development of future prediction and forecast models for pathogenic vibrios in market oysters. Objective 3. Probe the biology, host range, and infectivity of predatory bacteria to optimize their potential use as treatment against shellfish-borne vibrios. Objective 4. Develop novel and comprehensive methods for virus detection in shellfish that may also have potential for other foods. Objective 5: Identify novel in vitro propagation methods for human norovirus and hepatitis E virus. Objective 6: Evaluate inactivation technologies for virus-contaminated shellfish and other foods.
Under objective 1, a cocktail of phages and predatory bacteria will be formulated from isolates collected during surveys of Delaware Bay oysters. Cocktail effectiveness in eliminating V. parahaemolyticus (VP) from seawater will be tested followed by efficacy testing of the cocktail against VP in naturally-contaminated, market-size oysters. Under objective 2, a quantitative Halobacteriovorax (HBX) assay will be developed using a most probable number (MPN) based approach to quantify HBX in seawater, oysters and marine sediments. Positive tubes will be determined by plaque assay. Alternative, enzyme-based assays will also be explored. In the second phase of this objective, information will be collected on HBX and total and pathogenic VP abundances in oysters, seawater and sediments for the development of future prediction and forecast models for pathogenic vibrios in oysters. Phage abundances will also be monitored by plaque assay. The goal of objective 3 is to further our understanding of factors that affect the biology, host range and infectivity of predatory bacteria under various environmental conditions and how HBX impact pathogenic VP levels in oysters and their environment. Among questions to be answered are: whether HBX replicates within oyster gut or gill tissues; what is the generation time for HBX in VP; do HBX persist or die in the absence of host vibrios, do environmental conditions (temperature, salinity, or pH) affect HBX infection and replication within host cells, and what is the host range of HBX isolates. Under objective 4, metagenomics will be used to detect viruses in shellfish. The principal challenges and limitations will be sample preparation and sensitivity, so several virus extraction procedures will be investigated. All methods will be evaluated for purity and yield of virus RNA using shellfish samples seeded with surrogate viruses. Laboratory-spiked shellfish and wild shellfish impacted by sewage outfalls or from other areas prone to contamination will be evaluated. The goal of objective 5 is to identify novel in vitro propagation methods for human norovirus (HuNoV) and hepatitis E virus (HEV). Two established embryonic cell lines from zebrafish will be investigated for HuNoV replication. After incubation for up to 2 weeks, virus yields will be determined by RT-qPCR. The feasibility of a surrogate trout HEV assay will also be investigated as a potential model system for HEV inactivation. Trout HEV will also be evaluated in nonthermal virus inactivation studies. Other potential HEV cultivation techniques will be investigated to assess the infectivity and inactivation of genotype 3 zoonotic HEV including a 3-dimensional, microgravity culture system. Under objective 6, inactivation technologies for virus-contaminated shellfish will be evaluated including: high pressure processing (HPP) of frozen oysters to reduce or eliminate HuNoV; the use of X-rays with and without singlet oxygen enhancers to inactivate surrogates for HuNoV, hepatitis A virus and HEV; and targeted heating with infrared or radiofrequency to eliminate viruses and bacteria in specific shellfish tissues.
This is a newly approved NP108 project. Please see final annual report 8072-42000-081-00D for further information.